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Main sol. J266

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Internal Solution-ID: S3953

Equipment needed: Autoclave Anaerobic workbench
Compound Amount Unit Conc. [g/L] Conc. [mM]
Rumen fluid , clarified
(see below) 		300.00 ml - -
KH2PO4 0.30 g 0.3 2.204
K2HPO4 0.30 g 0.3 1.722
(NH4)2SO4 0.30 g 0.3 2.27
NaCl 0.60 g 0.6 10.267
MgSO4 x 7 H2O 0.13 g 0.13 0.527
CaCl2 x 2 H2O 8.00 mg 0.008 0.054
Trace minerals 10.00 ml - -
Trace vitamins 10.00 ml - -
FeSO4 x 7 H2O 2.00 mg 0.002 0.007
NaHCO3 2.00 g 2 23.808
Yeast extract
(BD-Difco) 		1.00 g 1 -
Trypticase peptone
(BD-BBL) 		1.00 g 1 -
Fatty acid mixture 20.00 ml - -
Resazurin 1.00 mg 1.0e-3 0.004
Na2S x 9 H2O 0.50 g 0.5 2.082
L-Cysteine HCl x H2O 0.50 g 0.5 -
Distilled water 660.00 ml - -
1 Mix ingredients, except NaHCO3, L-cysteine x HCl x H2O and Na2S x 9H2O, and adjust pH to 6.5. Bring to a boil for 5-10 sec, then, cool down under a stream of H2-CO2 (80:20, v/v) and add NaHCO3 to the medium. Dispense the medium into suitable culture vessels (e.g., 20 ml of the medium in 120 ml serum bottles) under a stream of H2-CO2 (80:20, v/v) and seal with butyl rubber stoppers. After autoclaving, stand the medium overnight. Separately autoclave cysteine x HCl and Na2S x 9H2O as 5% solutions, respectively, under a N2 atmosphere. Prior to inoculation, aseptically and anaerobically add the L-cysteine x HCl x H2O and Na2S x 9H2O solutions. Pressurize the inoculated bottles to 200 kPa H2-CO2 (80:20, v/v).
2 To prepare the clarified rumen fluid, preheat rumen content at 120C for 15 min. Use the supernatant after centrifuging at 25,000 x g for 15 min.
J266 METHANOMICROBIUM MOBILE MEDIUM